The Gene Disruption Project (GDP) has provided the research community with new genetic tools to support the functional analysis of the Drosophila genome and is responsible for generating 85% of the more than 15,000 transposable element (TE) insertion mutant stocks that are present in the Bloomington Drosophila Stock Center (BDSC). The newest stocks were generated using MiMIC, a TE that allows site-specific recombination-based tools for in vivo manipulation of the genome. We now propose to expand the GDP collection to increase its coverage and provide new methods for analyzing gene function. With the advent of the CRISPR gene editing technology, it is now technically feasible to make targeted modifications in user-defined locations rather than at random as is done with TEs. We propose to use CRISPR to introduce swappable insertion cassettes (SICs) into 5,000 genes missed by MiMIC. Three thousand genes that are evolutionarily conserved and are likely to be implicated in human disease will be prioritized. The remaining 2,000 will be selected based on the needs and interests of the community. In addition, we propose to generate protein trap alleles for 2,500 genes using existing MiMIC lines as well as new insertion lines developed in this proposal. These protein trap alleles will encode an in-frame GFP fusion and allow the determination of precise protein distribution using light and electron microscopy. They also allow efficient knockdown of mRNA transcript or protein product in specific cells or tissues using RNAi and deGradFP methodologies against the GFP tag. Furthermore, protein tagging allows purification strategies using nanobodies against GFP such as immunoprecipitation (IP) of proteins, chromatin IP for DNA-associated proteins, and IP-mass spectroscopy. New insertion and protein trap strains will be validated, and the supporting information submitted to public repositories such as GenBank and Flybase. Stocks generated by the GDP will be deposited at the BDSC for public distribution.